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. Author manuscript; available in PMC: 2020 Apr 18.
Published in final edited form as: Cell. 2019 Apr 4;177(3):766–781.e24. doi: 10.1016/j.cell.2019.02.009

Figure 6. The Arabidopsis ATG8-interacting PUX Proteins are Autophagy Receptors for Dysfunctional CDC48 Complexes.

Figure 6.

(A and B) PUX7 and CDC48a are degraded via autophagy upon nitrogen starvation. WT or autophagy-defective seedlings were starved for nitrogen for 16 hours, and release of free GFP or YFP from the GFP-PUX7 (panel (A)) or YFP-CDC48a (panel (B)) reporters was monitored by immunoblot analysis of total protein extracts with anti-GFP antibodies. Open and closed arrowheads locate fused and free GFP/YFP, respectively. Immunodetection of histone H3 was used to confirm near-equal protein loading.

(C and D) PUX7 and CDC48a are degraded via autophagy upon treatment with CB-5083. Seedlings were treated with or without 20 μM CB-5083 for 16 hours and GFP or YFP release was monitored as in panel (A).

(E and F) Inhibitor-induced autophagy of CDC48 requires PUX7, 8, 9 or 13. WT, atg7-2 or pux mutant seedlings were treated as in panel (D), and YFP release was monitored as in panel (B).

(G) Inhibited YFP-CDC48a accumulates in the vacuole in an autophagy- and PUX-dependent manner. Seedlings were treated as in panels (B) and (D), and imaged by confocal fluorescence microscopy. Scale bar, 10 μm. Nu, nucleus; Va, vacuole.

(H) CB-5083-induced vacuolar puncta containing YFP-CDC48a co-localize with the autophagic body marker mCherry-ATG8a. Seedlings were grown and analyzed as in panel (G).

(I) Homozygous pux-q plants are hypersensitive to CB-5083. WT or pux-q seedlings were grown for 10 days on GM medium containing either DMSO (control) or 2.5 μM CB-5083.

(J) Quantification of WT and pux-q seedling sensitivity to CB-5083; values represent the mean fresh weight (±SD) from three independent biological replicates.

(K and L) Mutations connected to human disease trigger PUX-dependent autophagy of Arabidopsis CDC48a. YFP-CDC48a variants were transiently expressed in protoplasts derived from WT, atg7-2 or pux-q plants, and YFP release was monitored as in panel (A).

(M) Mutations that trigger CDC48a degradation cluster within the N domain. The 3-dimensional structure of human CDC48 (p97; PDB file 5FTK) is shown, with residues identified in panel (K) highlighted in red.

(N) WT CDC48 is degraded by autophagy when associated with a mutant version. Seedlings expressing YFP-CDC48a were induced to express 6His-T7-CDC48a (either WT or DN mutant) in WT or atg7-2 backgrounds and harvested at the indicated times. YFP release was assayed as in panel (A), while immunodetection of the T7 tag confirmed transgene induction.